- Email: [email protected]
Neuropeptide-immunoreactive cells in human thymus
BRAIN,
BEHAVIOR,
AND
IMMUNITY
4, 189-197 (1990)
Neuropeptide-lmmunoreactive
Cells in Human Thymus
M. PIANTELLI, N. MAGGIANO, L. M. LAROCCA, R. RICCI, F. 0. RANELLETTI,” L. LAURIOLA, AND A. CAPELLI Istituti
di Anatomia Patologica e di *Istologia. Universitri Cattolica S. Cuore, 00168 Roma, Italy The outer cortex of the human thymus contains a one- to two-cell-thick layer that is immunoreactive with antisera against P-endorphin, (Leu)- and (Met)-enkephalin, bombesin, and substance P. The epithehal nature of these immunostained cells is revealed by immunoelectron microscopic studies showing the presence of desmosomal junctions. The presence of peptide-containing cells in the outer cortex, where the most immature and recently immigrated thymocytes are found, emphasizes the role of neuropeptides in regulating the microenvironment for T cell development. o IWO Academic press, hc.
INTRODUCTION
The immune and neuroendocrine systems appear able to communicate with each other by virtue of signal molecules (hormones) and receptors common to both systems (see Berczi, 1986, for a review). A large number of peptides, commonly regarded as capable of neurohormonal activity, have been found to have effects on, and sometimes also to occur in, cells of the imqune system (Angeletti & Hichey, 1987). In particular, it has been demonstrated that lymphocytes are able to produce and process not only proopiomelanocortin precursor but also ACTH, p-endorphin, and P-lipotrophin (Lolait, Lim, Toh, & Funder, 1984; Lolait, Clements, Markwick, Cheng, MacNally, Smith, & Funder, 1986; Smith, Harbour-McMenamin, & Blalock, 1985; Westly, Kleigs, Wong, & Yuen, 1986). In addition, endogenous opiates have been shown to modulate several immune functions such as antibody synthesis, lymphocyte mitogenesis, and natural killer cell activity (see Larsson, 1988, for a review). Furthermore, substance P has been found to increase the phagocytic activity of macrophages and polymorphonuclear leukocytes as well as protein and DNA synthesis in human T lymphocytes (Payan & Goetzl, 1985). Substance P and bombesin were also shown to influence lymphocyte migration through lymph nodes (Moore, 1984). Neuropeptides may also play an immunoregulatory role within the thymus. On this point, it has been reported that the human thymus contains measurable amounts of oxytocin and vasopressin (Geenen, Legros, Franchimont, Baudrikaye, Defense, & Bonvier, 1986; Geenen, Legros, & Franchimont, 1987). Moreover, immunohistochemical analysis has demonstrated the occurrence of both oxytocin- and vasopressin-like activity in thymic epithelial cells (Moll, Lane, Robert, Geenen, & Legros, 1988). Immunoreactive neurotensin, somatostatin, and substance P have also been found in chicken, rat, and guinea pig thymus (Sundler, Carraways, Hakanson, Alumets, & Dubois, 1979; Geppetti, Maggi, Zecchi-Orlandini, Santicioli, Meli, Frilli, Spillantini, & Amenta, 1987). In this paper we report the presence and distribution in the human thymus of a 189 0889-1591/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
190
PIANTELLI
ET
AL.
distinct population of epithelial cells immunoreactive and (Leu)-enkephalin, bombesin, and substance P. MATERIALS
with B-endorphin,
(Met)-
AND METHODS
Thymus fragments were obtained during open heart surgery from nine (1 month to 4 years old) infants and fixed in 0.4% p-benzoquinone in 0.01 M PBS, pH 7.3, for 1-4 h. Following fixation, the specimens were transferred to PBS containing 15% sucrose and 0.1% sodium azide. The tissue was then frozen for cryostat sectioning. Five-micrometer sections were mounted on slides coated with polyL-lysine (Sigma, Deisenhofen, West Germany), treated with 0.03% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity and incubated for 1 h with the following rabbit anti-peptide polyclonal antibodies at dilutions ranging from 1:400 to 1:800: anti-B-endorphin, anti-(Leu)- and anti(Met)-enkephalin, anti-bombesin, and anti-substance P (Amersham, England, and UCB Bioproducts, Belgium). Indirect immunostaining was achieved using both the PAP (Dako, Copenhagen, Denmark) (Sternberger, Hardy, Cuculis, & Meyer, 1970) and ABC (Vector, Burlington, MA) (Hsu, Raine, & Fanger, 1981) techniques. The peroxidase was developed with 3-amino-9-ethyl-carbazole following the procedure reported by Graham, Ludholm, and Karnovsky (1965). For electron microscopy small thymus fragments were cut on a Sorvall TC-2 sectioner, fixed in 4% paraformaldehyde for 2 h, and processed by the PAP method, using anti-peptide antibodies as previously reported (Lauriola, Michetti, Stolfi, Tallini, & Cocchia, 1984). After immunoreaction, the slices were postlixed in 1% 0~0, in 0.1 M PBS for 30 min, dehydrated in ethanol and propylene oxide, and embedded in Epon 812. Sections 8&90 nm in thickness were lightly counterstained with lead citrate and observed with a Philips EM 400. The antiserum to B-endorphin (UCB Bioproducts) was tested (immunoassay) by the manufacturer. The specificity of this antiserum, expressed as percentage of fixation (B/TxlOO) of hormone tracers ( 12’1) at I:200 dilution was 59 for B-endorphin and 1 for y-endorphin. No fixation was observed with ol-endorphin, Leu-enkephalin, LH-RH, TRH, and corticotropin-like intermediate peptide. Antibombesin antiserum partially cross-reacted with substance P. It was then afftnitypurified with substance P (Boehringer, Mannheim, West Germany) coupled to CNBr-activated Sepharose 4B (1 mg peptide/ml swollen bead). Anti-substance P antiserum tested by the manufacturer did not cross-react with bombesin. The specificity of the immunolabeling was tested by preabsorbing antisera with their homologous synthetic antigens (loo-fold w/w excess) (Boehringer Mannheim, West Germany). Following this procedure, immunoreaction was abolished. Positive controls consisted of hypothalamic tissue obtained from two postmortem cases. Negative controls consisted of normal rabbit serum or anti-thyroglobulin rabbit antibodies (Dako Patts a/s, Denmark) used in place of the primary antiserum. Negative controls were also performed by omitting either the second antiserum or the PAP or ABC complexes. All anti-peptide antisera were unreactive when tested on normal human skin and on squamous cell carcinoma samples. To avoid nonspecific immunocytochemical reactions, all antisera were diluted in 2 mg/ml poly-L-lysine containing buffer as suggested by Scopsi, Wang, and Larsson
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
191
(1986). Single cell suspensions of thymic lymphocytes were obtained and immunostained as previously reported (Maggiano, Larocca, Piantelli, Acute, & Musiani, 1987). RESULTS
All human thymuses studied showed a normal histology for the respective age. When immunohistochemistry was performed, a common pattern of immunostaining was observed with all peptide antisera (Figs. 1 and 2). In the sections from all thymuses a rim of immunoreactive cells was observed in the outer, subcapsular cortex. The vast majority of the remaining cortical cells were nonreactive but a few individual scattered positive cells were also found in this area as well as in the medulla. The immunolabeled cells constituted an apparently discontinuous layer one to two cells thick beneath the capsular fibrous tissue. These cells were mainly irregular in shape with numerous cytoplasmic projections. In the connective tissue septa some round, lightly stained cells, mainly present in the blood vessels, were easily recognized as erythrocytes that had partially escaped the procedures
FIG. 1. Consecutive sections of normal thymus treated with anti-P-endorphin and anti-bombesin antiserum. Note the immunolabeled cells mainly located at the periphery of the lobules. X 120.
FIG. 2. At higher magnification the morphology and peculiar location of the immunolabeled cells is apparent. Sections of normal thymus were treated with anti-f3-endorphin (a), anti-(Leu)-enkephalin, (b), and anti-(Met)-enkephalin (c) antiserum. x200. The location of the immunolabeled cells is apparent after treatment with anti-substance P antiserum (d). x96. 192
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
193
to block endogenous peroxidase activity. Thymic lymphocytes obtained from normal thymus did not react with any of the anti-peptide antisera. Immunoelectron microscopic studies were also performed on tissue fragments from five different glands in order to better characterize the immunoreactive cells. After immunostaining, the immunolabeled cells were morphologically heterogeneous and could be classified according to the criteria reported by van de Wijngaert et al. (1984). Figure 3 shows more deeply located type 2/3 epithelial cells that are also occasionally shown to be immunoreactive in Figs. 2a and 2c. In particular, in Fig. 3a, a type 2 “pale” cell with round euchromatic nucleus, short profiles of RER, and long cytoplasmic processes is shown. Figure 3b shows an immunoreactive “intermediate” cell type with irregularly shaped nucleus and prominent nucleolus. The cells appeared frequently to be linked to each other and to adjacent unlabeled epithelia by desmosomes. Moreover they were found to give off long slender cytoplasmic processes which adhered to adjacent lymphoid cells. Immunolabeled cells located beneath the capsule, recognizable as type 1 cells according to the criteria proposed by van de Wijngaert et al. (1984), appeared elongated with abundant RER and heterochromatic nucleus (Fig. 4). No labeled cells at either the light or the electron microscopic level were found in negative control sections (not shown). DISCUSSION
In this paper we report the presence in the human thymus of a distinct cell subset immunoreactive for neuropeptides. The pattern of distribution of the immunolabeled cells was characteristic, most being located in a narrow subcapsular area of the cortex with only a few positive cells in the remaining cortex and medulla. The epithelial nature of these thymic cells is revealed by the presence of desmosomes between them. The peculiar location of these neuropeptidecontaining cells agrees with previous findings (Haynes, Shimizu, & Eisenbarth, 1983; Gaudecker, Steinmann, Hansmann, Harpprecht, Milicevic, & MtillerErmelink, 1986). Epithelial cells in the subcapsular cortex of normal thymus are labeled by the A2B5 monoclonal antibody which reacts with a complex neuronal ganglioside expressed on the cell surface of neurons, neural crest-derived cells, and peptide-secreting endocrine cells. An abundant occurrence of oxytocin and vasopressin in the subcapsular cortex and in the medulla has been shown (Moll et al. 1988) and the detection of mRNA for these peptides in the thymuses of children indicates an in situ synthesis for both oxytocin and vasopressin (Geenen et al., 1987). Our data support the hypothesis of a local expression of B-endorphin and (Leu)and (Met)-enkephalin. However, since these neuropeptides may be part of larger precursor molecules (i.e., proopiomelanocortin, preproenkephalin), we cannot exclude the possibility that the antibodies used, although specific, are actually detecting neuropeptides as a part of their precursor molecules. Whatever the case, it is interesting to note that cells positive for bombesin and substance P, both unrelated to the opioid family, exhibited a similiar pattern of intrathymic distribution. In addition, the measurable amounts of substance P-like immunoreactivity present in the thymus of rats or guinea pigs, when tested by a HPLC system,
194
PIANTELLI
ET
AL.
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
FIG. 4. Normal thymus treated with anti-R-endorphin antiserum. Three elongated immunolabeled cells with etherochromatic nuclei and abundant RER are located beneath the capsule. x5700.
eluted as authentic substance P (Geppetti et al., 1987). However, the present data do not rule out the possibility that the peptides investigated are produced by different cell types with similar topographical location. According to previous ultrastructural reports (Nabarra & Andrianarison, 1987), we have observed that secretory granules are extremely rare in thymic subcapsular epithelia. This finding may be explained by recent data supporting the existence in the thymus of a secretory system different from the usual neuroendocrine model, based on “clear vacuoles” rather than on the classical secretory granules (Nabarra & Andrianarison, 1987). Moreover, the general appearance of the immunoreactive cells, which give off slender cytoplasmic processes adhering to surrounding lymphoid cells, is that of a paracrine cell. A direct connection with the nervous system is established by both ortho- and para-sympathetic fibers innervating the thymic subcortical area (Bulloch, 1985; Felten, Felten, Carlson, Olschowka, & Livnat, 1985). Like other neurosecretory cells, thymic peptide containing cells could convert a neuronal signal into a peptide secretion. It is noteworthy that in mice the most immature intrathymic lymphocytes with T cell progenitor activity in adoptive transfer experiments (Fowlkes, Edison, FIG. 3. Normal thymus treated with anti-@-endorphin antiserum. (a) Immunoreaction product is confined to the cytoplasmic matrix of an epithelial cell which sends cytoplasmic processes between adjacent lymphocytes (arrowhead). Note a desmosome at the periphery of the immunolabeled cell (arrow). x8100. (b) A subcapsular immunostained cell with elongated cytoplasmic processes (arrowhead) after treatment with anti-(Met)-enkephalin antiserum. X8100.
196
PIANTELLI
ET AL.
Mathieson, & Chused, 1985) were found by histochemistry to be confined to the subcapsular area of the thymus (Fan-, Anderson, Braddy, & Mejino, 1988). In the human thymus, the lymphoid components of the subcapsular cortical region are the large TdT+ blasts which do not express cortical (CDl-) and medullary (CD3-) cell markers (Janossy, Bofill, Trejdosiewicz, W~&U,& Chilosi, 1986; Piantelli, Larocca, Aiello, Maggiano, Carbone, Ranelletti, & M siani, 1986). The presence of peptide-containing cells in the subcapsular area sk&5 ,.sts that these peptides may act at an early stage of intrathymic T cell differentiation. Neuropeptides are also reported to promote growth of several cell types including fibroblasts, chondrocytes, bronchial and gastric epithelial cells, and splenic lymphocytes (for a review see Zachary, Woll, & Ro?engurt, 1987). From the above data, it seems possible that in the thymus neuropeptides may represent growth and regulatory signals for both immature and recently immigrated lymphoblasts and/or surrounding epithelial cells. ACKNOWLEDGMENTS This work was partially supported by grants from C.N.R. (Progetto Finalizzato) and M.P.I. (40% and 60%). R.R. is a recipient of a fellowship from Sclavo S.p.A. The authors thank Mr Alessandro Rinelli for helpful technical assistance and Mr Paolo Baldassani for editorial assistance.
REFERENCES Angeletti, R. H., & Hichey, W. F. (1987). Neuroendocrine cells within immune tissues. Ann. N. Y. Acad. Sci. 496, 78-84. Berczi, I. (1986). Pituitary function and immunity. CRC Press: Boca Raton, FL. Bulloch, K. (1985). Neuroanatomy of lymphoid tissue: A review. In R. Guillemin, M. Cohn, & T. Melnechuk (Eds.), Neural modulation ofimmuniry pp. 111-141. Raven Press: New York. Farr, A. G., Anderson, S. K., Braddy, S. C., & Mejino, J. L. V. (1988). Selective binding of Dolichos bifloris agglutinin to L3T4-, LYT-2-thymocytes. Expression of terminal a-linked Nacetyl-D-galactosamine residues defines a subpopulation of fetal and adult murine thymocytes. J. Immunol. 140, 1014-1021. Felten, D. L., Felten, S. Y., Carlson, S. L., Olschowka, J. A., & Livnat, S. (1985). Noradrenergic and peptidergic innervation of lymphoid tissue. .I. Immunol. 135, 755s-765s. Fowlkes, B. J., Edison, L., Mathieson, B. J., & Chused, T. M. (1985). Early T lymphocytes: Differentiation in vivo of intrathymic precursor cells. J. Exp. Med. 162, 802-822. Gaudecker, B. V., Steinmann, G. G., Hansmann, M.-L., Harpprecht, J., Milicevic, N. M., & MiillerErmelink, H.-K. (1986). Immunohistochemical characterization of the thymic microenvironment. A light-microscopic and ultrastructural immunocytochemical study. Cell Tissue Res. 244, 403412. Geenen, V., Legros, J. J., Franchimont, P., Baudrikaye, M., Defense, M.-P., & Bonvier, J. (1986). The neuroendocrine thymus: Coexistence of oxytocin and neurophysin in the huyman thymus. Science 232, 508-S 11. Geenen, V., Legros, J. J., & Franchimont, P. (1987). The thymus as a neuroendocrine organ: Synthesis of vasopressin and oxytocin in human thymic epithelium. Ann. N. Y. Acad. Sci. 4%, 56-66. Geppetti, P., Maggi, C. A., Zecchi-Orlandini, S., Santicioli, P., Meli, A., Frilli, S., Spillantini, M. G., & Amenta, F. (1987). Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus. Regul. Pep?. 18, 321-329. Graham, R. C. Jr, Ludholm, U., & Kartnovsky, M. J. (1965). Cytochemical demonstration of peroxidase activity with 3-amino 9-ethyl carbazole. J. Histochem. Cytochem. 13, 150-152. Hsu, S. M., Raine, L., & Fanger, H. (1981). The use of antiavidin antibody and avidin-biotinperoxidase complex in immunoperoxidase techniques. Amer. J. Clin. Pathol. 75, 816-821.
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
197
Haynes, B. F., Shimizu, K., & Eisenbarth, G. S. (1983). Identification of human and rodent thymus epithelium using tetanus toxin and monoclonal antibody A2B5. J. C/in. Invest. 71, 9-14. Janossy, G., Bofill, M., Trejdosiewicz, L. K., Villcox, H. N. A., & Chilosi, M. (1986). Cellular differentiation of lymphoid subpopulations and their microenvironments in the human thymus. In H. K. Mtiller-Hermelink (ed.), Current topics in Pathology, Vol. 75, pp. 89-125. Springer: Berlin/ Heidelberg/New York. Larsson, L.-I. (1988). Regulatory peptides and amines during ontogeny and in non-endocrine cancers: Occurrence and possible functional significance. In Progress in Histochemistry and Cytochemistry, Vol. 17, No. 4, pp. 157-169. Gustav Fisher Verlag: Stuttgart/New York. Lauriola, L., Michetti, F., Stolti, V. M., Tallini, G., & Cocchia, D. (1984). Detection by S-100 immunolabelling of interdigitating reticulum cells in human thymomas. Virchow Arch. 45, 187195. Lolait, S. J., Clements, J. A., Markwick, A. J., Cheng, C., MacNally, M., Smith, A. I., & Funder, J. W. (1986). Pro-opiomelanocortin messenger ribonucleic acid and post translational processing of P-endorphin in spleen macrophages. J. Clin. Invest. 77, 17761779. Lolait, S. J., Lim, A. T. W., Toh, B. H., & Funder, J. W. (1984). Immunoreactive B-endorphin in a subpopulation of mouse spleen macrophages. J. C/in. Invest. 73, 277-280. Maggiano, N., Larocca, L. M., Piantelli, M., Acute, O., & Musiani, P. (1987). Expression of T cell receptor a and 8 subunits in human thymocytes. An immunocytologic study. J. Zmmunol. 139, 1438-1445. Moll, U. M., Lane, B. L., Robert, F., Geenen, V., & Legros J.-J. (1988). Abundant occurrence of oxytocin-, vasopressin-, and neurophysin-like peptides in epithelial cells. Histochemistry 89, 385-390. Moore, T. C. (1984). Modification of lymphocyte traflic by vasoactive neurotransmitter substances. Immunology 52, 511-518. Nabarra, B., & Andrianarison, I. (1987). Pattern of secretion in thymic epithelial cells: Ultrastructural studies of the effect of blockage at various levels. Cell Tissue Res. 249, 171-178. Payan, D. G., & Goetzl, E. J. (1985). Modulation of lymphocyte function by sensory neuropeptides. J. Zmmunol. 135, 783s-786s. Piantelli, M., Larocca, L. M., Aiello. F. B., Maggiano, N., Carbone, A., Ranelletti, F. 0.. & Musiani, P. (1986). Proliferation of phenotypically immature human thymocytes with and without interleukin 2 receptors. J. Zmmunol. 136, 3204-3210. Scopsi, L., Wang, B.-L., & Larsson, L.-I. (1986). Nonspecific immunocytochemical reactions with certain neurohormonal peptides and basic peptide sequences. J. Histochem. Cytochem. 34, 14691475. Smith, E. M., Harbour-McMenamin, D., & Blalock, J. E. (1985). Lymphocyte production of endorphins and endorphin-mediated immunoregulatory activity. J. Zmmunol. 135, 779s-782s. Sternberger, L. A., Hardy, P. H. Jr, Cuculis, J. J., & Meyer, H. G. (1970). The unlabeled antibodyenzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333. Sundler, F., Carraways, R. E., Hakanson, R., Alumets, J., & Dubois M. P. (1979). Immunoreactive neurotensin and somatostatin in the chicken thymus. Cell Tissue Res. 194, 367-376. Westly, H. J., Kleigs, A. J., Kelley, K. W., Wong, P. K. Y., & Yuen, P.-H. (1986). Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J. Exp. Med. 163, 1589-1594. Zachary, I., Woll, P. J., & Rozengurt, E. (1987). A role for neuropeptides in the control of cell proliferation. Dev. Viol. 124, 295-308.
BEHAVIOR,
AND
IMMUNITY
4, 189-197 (1990)
Neuropeptide-lmmunoreactive
Cells in Human Thymus
M. PIANTELLI, N. MAGGIANO, L. M. LAROCCA, R. RICCI, F. 0. RANELLETTI,” L. LAURIOLA, AND A. CAPELLI Istituti
di Anatomia Patologica e di *Istologia. Universitri Cattolica S. Cuore, 00168 Roma, Italy The outer cortex of the human thymus contains a one- to two-cell-thick layer that is immunoreactive with antisera against P-endorphin, (Leu)- and (Met)-enkephalin, bombesin, and substance P. The epithehal nature of these immunostained cells is revealed by immunoelectron microscopic studies showing the presence of desmosomal junctions. The presence of peptide-containing cells in the outer cortex, where the most immature and recently immigrated thymocytes are found, emphasizes the role of neuropeptides in regulating the microenvironment for T cell development. o IWO Academic press, hc.
INTRODUCTION
The immune and neuroendocrine systems appear able to communicate with each other by virtue of signal molecules (hormones) and receptors common to both systems (see Berczi, 1986, for a review). A large number of peptides, commonly regarded as capable of neurohormonal activity, have been found to have effects on, and sometimes also to occur in, cells of the imqune system (Angeletti & Hichey, 1987). In particular, it has been demonstrated that lymphocytes are able to produce and process not only proopiomelanocortin precursor but also ACTH, p-endorphin, and P-lipotrophin (Lolait, Lim, Toh, & Funder, 1984; Lolait, Clements, Markwick, Cheng, MacNally, Smith, & Funder, 1986; Smith, Harbour-McMenamin, & Blalock, 1985; Westly, Kleigs, Wong, & Yuen, 1986). In addition, endogenous opiates have been shown to modulate several immune functions such as antibody synthesis, lymphocyte mitogenesis, and natural killer cell activity (see Larsson, 1988, for a review). Furthermore, substance P has been found to increase the phagocytic activity of macrophages and polymorphonuclear leukocytes as well as protein and DNA synthesis in human T lymphocytes (Payan & Goetzl, 1985). Substance P and bombesin were also shown to influence lymphocyte migration through lymph nodes (Moore, 1984). Neuropeptides may also play an immunoregulatory role within the thymus. On this point, it has been reported that the human thymus contains measurable amounts of oxytocin and vasopressin (Geenen, Legros, Franchimont, Baudrikaye, Defense, & Bonvier, 1986; Geenen, Legros, & Franchimont, 1987). Moreover, immunohistochemical analysis has demonstrated the occurrence of both oxytocin- and vasopressin-like activity in thymic epithelial cells (Moll, Lane, Robert, Geenen, & Legros, 1988). Immunoreactive neurotensin, somatostatin, and substance P have also been found in chicken, rat, and guinea pig thymus (Sundler, Carraways, Hakanson, Alumets, & Dubois, 1979; Geppetti, Maggi, Zecchi-Orlandini, Santicioli, Meli, Frilli, Spillantini, & Amenta, 1987). In this paper we report the presence and distribution in the human thymus of a 189 0889-1591/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
190
PIANTELLI
ET
AL.
distinct population of epithelial cells immunoreactive and (Leu)-enkephalin, bombesin, and substance P. MATERIALS
with B-endorphin,
(Met)-
AND METHODS
Thymus fragments were obtained during open heart surgery from nine (1 month to 4 years old) infants and fixed in 0.4% p-benzoquinone in 0.01 M PBS, pH 7.3, for 1-4 h. Following fixation, the specimens were transferred to PBS containing 15% sucrose and 0.1% sodium azide. The tissue was then frozen for cryostat sectioning. Five-micrometer sections were mounted on slides coated with polyL-lysine (Sigma, Deisenhofen, West Germany), treated with 0.03% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity and incubated for 1 h with the following rabbit anti-peptide polyclonal antibodies at dilutions ranging from 1:400 to 1:800: anti-B-endorphin, anti-(Leu)- and anti(Met)-enkephalin, anti-bombesin, and anti-substance P (Amersham, England, and UCB Bioproducts, Belgium). Indirect immunostaining was achieved using both the PAP (Dako, Copenhagen, Denmark) (Sternberger, Hardy, Cuculis, & Meyer, 1970) and ABC (Vector, Burlington, MA) (Hsu, Raine, & Fanger, 1981) techniques. The peroxidase was developed with 3-amino-9-ethyl-carbazole following the procedure reported by Graham, Ludholm, and Karnovsky (1965). For electron microscopy small thymus fragments were cut on a Sorvall TC-2 sectioner, fixed in 4% paraformaldehyde for 2 h, and processed by the PAP method, using anti-peptide antibodies as previously reported (Lauriola, Michetti, Stolfi, Tallini, & Cocchia, 1984). After immunoreaction, the slices were postlixed in 1% 0~0, in 0.1 M PBS for 30 min, dehydrated in ethanol and propylene oxide, and embedded in Epon 812. Sections 8&90 nm in thickness were lightly counterstained with lead citrate and observed with a Philips EM 400. The antiserum to B-endorphin (UCB Bioproducts) was tested (immunoassay) by the manufacturer. The specificity of this antiserum, expressed as percentage of fixation (B/TxlOO) of hormone tracers ( 12’1) at I:200 dilution was 59 for B-endorphin and 1 for y-endorphin. No fixation was observed with ol-endorphin, Leu-enkephalin, LH-RH, TRH, and corticotropin-like intermediate peptide. Antibombesin antiserum partially cross-reacted with substance P. It was then afftnitypurified with substance P (Boehringer, Mannheim, West Germany) coupled to CNBr-activated Sepharose 4B (1 mg peptide/ml swollen bead). Anti-substance P antiserum tested by the manufacturer did not cross-react with bombesin. The specificity of the immunolabeling was tested by preabsorbing antisera with their homologous synthetic antigens (loo-fold w/w excess) (Boehringer Mannheim, West Germany). Following this procedure, immunoreaction was abolished. Positive controls consisted of hypothalamic tissue obtained from two postmortem cases. Negative controls consisted of normal rabbit serum or anti-thyroglobulin rabbit antibodies (Dako Patts a/s, Denmark) used in place of the primary antiserum. Negative controls were also performed by omitting either the second antiserum or the PAP or ABC complexes. All anti-peptide antisera were unreactive when tested on normal human skin and on squamous cell carcinoma samples. To avoid nonspecific immunocytochemical reactions, all antisera were diluted in 2 mg/ml poly-L-lysine containing buffer as suggested by Scopsi, Wang, and Larsson
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
191
(1986). Single cell suspensions of thymic lymphocytes were obtained and immunostained as previously reported (Maggiano, Larocca, Piantelli, Acute, & Musiani, 1987). RESULTS
All human thymuses studied showed a normal histology for the respective age. When immunohistochemistry was performed, a common pattern of immunostaining was observed with all peptide antisera (Figs. 1 and 2). In the sections from all thymuses a rim of immunoreactive cells was observed in the outer, subcapsular cortex. The vast majority of the remaining cortical cells were nonreactive but a few individual scattered positive cells were also found in this area as well as in the medulla. The immunolabeled cells constituted an apparently discontinuous layer one to two cells thick beneath the capsular fibrous tissue. These cells were mainly irregular in shape with numerous cytoplasmic projections. In the connective tissue septa some round, lightly stained cells, mainly present in the blood vessels, were easily recognized as erythrocytes that had partially escaped the procedures
FIG. 1. Consecutive sections of normal thymus treated with anti-P-endorphin and anti-bombesin antiserum. Note the immunolabeled cells mainly located at the periphery of the lobules. X 120.
FIG. 2. At higher magnification the morphology and peculiar location of the immunolabeled cells is apparent. Sections of normal thymus were treated with anti-f3-endorphin (a), anti-(Leu)-enkephalin, (b), and anti-(Met)-enkephalin (c) antiserum. x200. The location of the immunolabeled cells is apparent after treatment with anti-substance P antiserum (d). x96. 192
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
193
to block endogenous peroxidase activity. Thymic lymphocytes obtained from normal thymus did not react with any of the anti-peptide antisera. Immunoelectron microscopic studies were also performed on tissue fragments from five different glands in order to better characterize the immunoreactive cells. After immunostaining, the immunolabeled cells were morphologically heterogeneous and could be classified according to the criteria reported by van de Wijngaert et al. (1984). Figure 3 shows more deeply located type 2/3 epithelial cells that are also occasionally shown to be immunoreactive in Figs. 2a and 2c. In particular, in Fig. 3a, a type 2 “pale” cell with round euchromatic nucleus, short profiles of RER, and long cytoplasmic processes is shown. Figure 3b shows an immunoreactive “intermediate” cell type with irregularly shaped nucleus and prominent nucleolus. The cells appeared frequently to be linked to each other and to adjacent unlabeled epithelia by desmosomes. Moreover they were found to give off long slender cytoplasmic processes which adhered to adjacent lymphoid cells. Immunolabeled cells located beneath the capsule, recognizable as type 1 cells according to the criteria proposed by van de Wijngaert et al. (1984), appeared elongated with abundant RER and heterochromatic nucleus (Fig. 4). No labeled cells at either the light or the electron microscopic level were found in negative control sections (not shown). DISCUSSION
In this paper we report the presence in the human thymus of a distinct cell subset immunoreactive for neuropeptides. The pattern of distribution of the immunolabeled cells was characteristic, most being located in a narrow subcapsular area of the cortex with only a few positive cells in the remaining cortex and medulla. The epithelial nature of these thymic cells is revealed by the presence of desmosomes between them. The peculiar location of these neuropeptidecontaining cells agrees with previous findings (Haynes, Shimizu, & Eisenbarth, 1983; Gaudecker, Steinmann, Hansmann, Harpprecht, Milicevic, & MtillerErmelink, 1986). Epithelial cells in the subcapsular cortex of normal thymus are labeled by the A2B5 monoclonal antibody which reacts with a complex neuronal ganglioside expressed on the cell surface of neurons, neural crest-derived cells, and peptide-secreting endocrine cells. An abundant occurrence of oxytocin and vasopressin in the subcapsular cortex and in the medulla has been shown (Moll et al. 1988) and the detection of mRNA for these peptides in the thymuses of children indicates an in situ synthesis for both oxytocin and vasopressin (Geenen et al., 1987). Our data support the hypothesis of a local expression of B-endorphin and (Leu)and (Met)-enkephalin. However, since these neuropeptides may be part of larger precursor molecules (i.e., proopiomelanocortin, preproenkephalin), we cannot exclude the possibility that the antibodies used, although specific, are actually detecting neuropeptides as a part of their precursor molecules. Whatever the case, it is interesting to note that cells positive for bombesin and substance P, both unrelated to the opioid family, exhibited a similiar pattern of intrathymic distribution. In addition, the measurable amounts of substance P-like immunoreactivity present in the thymus of rats or guinea pigs, when tested by a HPLC system,
194
PIANTELLI
ET
AL.
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
FIG. 4. Normal thymus treated with anti-R-endorphin antiserum. Three elongated immunolabeled cells with etherochromatic nuclei and abundant RER are located beneath the capsule. x5700.
eluted as authentic substance P (Geppetti et al., 1987). However, the present data do not rule out the possibility that the peptides investigated are produced by different cell types with similar topographical location. According to previous ultrastructural reports (Nabarra & Andrianarison, 1987), we have observed that secretory granules are extremely rare in thymic subcapsular epithelia. This finding may be explained by recent data supporting the existence in the thymus of a secretory system different from the usual neuroendocrine model, based on “clear vacuoles” rather than on the classical secretory granules (Nabarra & Andrianarison, 1987). Moreover, the general appearance of the immunoreactive cells, which give off slender cytoplasmic processes adhering to surrounding lymphoid cells, is that of a paracrine cell. A direct connection with the nervous system is established by both ortho- and para-sympathetic fibers innervating the thymic subcortical area (Bulloch, 1985; Felten, Felten, Carlson, Olschowka, & Livnat, 1985). Like other neurosecretory cells, thymic peptide containing cells could convert a neuronal signal into a peptide secretion. It is noteworthy that in mice the most immature intrathymic lymphocytes with T cell progenitor activity in adoptive transfer experiments (Fowlkes, Edison, FIG. 3. Normal thymus treated with anti-@-endorphin antiserum. (a) Immunoreaction product is confined to the cytoplasmic matrix of an epithelial cell which sends cytoplasmic processes between adjacent lymphocytes (arrowhead). Note a desmosome at the periphery of the immunolabeled cell (arrow). x8100. (b) A subcapsular immunostained cell with elongated cytoplasmic processes (arrowhead) after treatment with anti-(Met)-enkephalin antiserum. X8100.
196
PIANTELLI
ET AL.
Mathieson, & Chused, 1985) were found by histochemistry to be confined to the subcapsular area of the thymus (Fan-, Anderson, Braddy, & Mejino, 1988). In the human thymus, the lymphoid components of the subcapsular cortical region are the large TdT+ blasts which do not express cortical (CDl-) and medullary (CD3-) cell markers (Janossy, Bofill, Trejdosiewicz, W~&U,& Chilosi, 1986; Piantelli, Larocca, Aiello, Maggiano, Carbone, Ranelletti, & M siani, 1986). The presence of peptide-containing cells in the subcapsular area sk&5 ,.sts that these peptides may act at an early stage of intrathymic T cell differentiation. Neuropeptides are also reported to promote growth of several cell types including fibroblasts, chondrocytes, bronchial and gastric epithelial cells, and splenic lymphocytes (for a review see Zachary, Woll, & Ro?engurt, 1987). From the above data, it seems possible that in the thymus neuropeptides may represent growth and regulatory signals for both immature and recently immigrated lymphoblasts and/or surrounding epithelial cells. ACKNOWLEDGMENTS This work was partially supported by grants from C.N.R. (Progetto Finalizzato) and M.P.I. (40% and 60%). R.R. is a recipient of a fellowship from Sclavo S.p.A. The authors thank Mr Alessandro Rinelli for helpful technical assistance and Mr Paolo Baldassani for editorial assistance.
REFERENCES Angeletti, R. H., & Hichey, W. F. (1987). Neuroendocrine cells within immune tissues. Ann. N. Y. Acad. Sci. 496, 78-84. Berczi, I. (1986). Pituitary function and immunity. CRC Press: Boca Raton, FL. Bulloch, K. (1985). Neuroanatomy of lymphoid tissue: A review. In R. Guillemin, M. Cohn, & T. Melnechuk (Eds.), Neural modulation ofimmuniry pp. 111-141. Raven Press: New York. Farr, A. G., Anderson, S. K., Braddy, S. C., & Mejino, J. L. V. (1988). Selective binding of Dolichos bifloris agglutinin to L3T4-, LYT-2-thymocytes. Expression of terminal a-linked Nacetyl-D-galactosamine residues defines a subpopulation of fetal and adult murine thymocytes. J. Immunol. 140, 1014-1021. Felten, D. L., Felten, S. Y., Carlson, S. L., Olschowka, J. A., & Livnat, S. (1985). Noradrenergic and peptidergic innervation of lymphoid tissue. .I. Immunol. 135, 755s-765s. Fowlkes, B. J., Edison, L., Mathieson, B. J., & Chused, T. M. (1985). Early T lymphocytes: Differentiation in vivo of intrathymic precursor cells. J. Exp. Med. 162, 802-822. Gaudecker, B. V., Steinmann, G. G., Hansmann, M.-L., Harpprecht, J., Milicevic, N. M., & MiillerErmelink, H.-K. (1986). Immunohistochemical characterization of the thymic microenvironment. A light-microscopic and ultrastructural immunocytochemical study. Cell Tissue Res. 244, 403412. Geenen, V., Legros, J. J., Franchimont, P., Baudrikaye, M., Defense, M.-P., & Bonvier, J. (1986). The neuroendocrine thymus: Coexistence of oxytocin and neurophysin in the huyman thymus. Science 232, 508-S 11. Geenen, V., Legros, J. J., & Franchimont, P. (1987). The thymus as a neuroendocrine organ: Synthesis of vasopressin and oxytocin in human thymic epithelium. Ann. N. Y. Acad. Sci. 4%, 56-66. Geppetti, P., Maggi, C. A., Zecchi-Orlandini, S., Santicioli, P., Meli, A., Frilli, S., Spillantini, M. G., & Amenta, F. (1987). Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus. Regul. Pep?. 18, 321-329. Graham, R. C. Jr, Ludholm, U., & Kartnovsky, M. J. (1965). Cytochemical demonstration of peroxidase activity with 3-amino 9-ethyl carbazole. J. Histochem. Cytochem. 13, 150-152. Hsu, S. M., Raine, L., & Fanger, H. (1981). The use of antiavidin antibody and avidin-biotinperoxidase complex in immunoperoxidase techniques. Amer. J. Clin. Pathol. 75, 816-821.
PEPTIDE-IMMUNOREACTIVE
THYMIC
CELLS
197
Haynes, B. F., Shimizu, K., & Eisenbarth, G. S. (1983). Identification of human and rodent thymus epithelium using tetanus toxin and monoclonal antibody A2B5. J. C/in. Invest. 71, 9-14. Janossy, G., Bofill, M., Trejdosiewicz, L. K., Villcox, H. N. A., & Chilosi, M. (1986). Cellular differentiation of lymphoid subpopulations and their microenvironments in the human thymus. In H. K. Mtiller-Hermelink (ed.), Current topics in Pathology, Vol. 75, pp. 89-125. Springer: Berlin/ Heidelberg/New York. Larsson, L.-I. (1988). Regulatory peptides and amines during ontogeny and in non-endocrine cancers: Occurrence and possible functional significance. In Progress in Histochemistry and Cytochemistry, Vol. 17, No. 4, pp. 157-169. Gustav Fisher Verlag: Stuttgart/New York. Lauriola, L., Michetti, F., Stolti, V. M., Tallini, G., & Cocchia, D. (1984). Detection by S-100 immunolabelling of interdigitating reticulum cells in human thymomas. Virchow Arch. 45, 187195. Lolait, S. J., Clements, J. A., Markwick, A. J., Cheng, C., MacNally, M., Smith, A. I., & Funder, J. W. (1986). Pro-opiomelanocortin messenger ribonucleic acid and post translational processing of P-endorphin in spleen macrophages. J. Clin. Invest. 77, 17761779. Lolait, S. J., Lim, A. T. W., Toh, B. H., & Funder, J. W. (1984). Immunoreactive B-endorphin in a subpopulation of mouse spleen macrophages. J. C/in. Invest. 73, 277-280. Maggiano, N., Larocca, L. M., Piantelli, M., Acute, O., & Musiani, P. (1987). Expression of T cell receptor a and 8 subunits in human thymocytes. An immunocytologic study. J. Zmmunol. 139, 1438-1445. Moll, U. M., Lane, B. L., Robert, F., Geenen, V., & Legros J.-J. (1988). Abundant occurrence of oxytocin-, vasopressin-, and neurophysin-like peptides in epithelial cells. Histochemistry 89, 385-390. Moore, T. C. (1984). Modification of lymphocyte traflic by vasoactive neurotransmitter substances. Immunology 52, 511-518. Nabarra, B., & Andrianarison, I. (1987). Pattern of secretion in thymic epithelial cells: Ultrastructural studies of the effect of blockage at various levels. Cell Tissue Res. 249, 171-178. Payan, D. G., & Goetzl, E. J. (1985). Modulation of lymphocyte function by sensory neuropeptides. J. Zmmunol. 135, 783s-786s. Piantelli, M., Larocca, L. M., Aiello. F. B., Maggiano, N., Carbone, A., Ranelletti, F. 0.. & Musiani, P. (1986). Proliferation of phenotypically immature human thymocytes with and without interleukin 2 receptors. J. Zmmunol. 136, 3204-3210. Scopsi, L., Wang, B.-L., & Larsson, L.-I. (1986). Nonspecific immunocytochemical reactions with certain neurohormonal peptides and basic peptide sequences. J. Histochem. Cytochem. 34, 14691475. Smith, E. M., Harbour-McMenamin, D., & Blalock, J. E. (1985). Lymphocyte production of endorphins and endorphin-mediated immunoregulatory activity. J. Zmmunol. 135, 779s-782s. Sternberger, L. A., Hardy, P. H. Jr, Cuculis, J. J., & Meyer, H. G. (1970). The unlabeled antibodyenzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333. Sundler, F., Carraways, R. E., Hakanson, R., Alumets, J., & Dubois M. P. (1979). Immunoreactive neurotensin and somatostatin in the chicken thymus. Cell Tissue Res. 194, 367-376. Westly, H. J., Kleigs, A. J., Kelley, K. W., Wong, P. K. Y., & Yuen, P.-H. (1986). Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J. Exp. Med. 163, 1589-1594. Zachary, I., Woll, P. J., & Rozengurt, E. (1987). A role for neuropeptides in the control of cell proliferation. Dev. Viol. 124, 295-308.